Expression of 1-aminocyclopropane-1-carboxylate (ACC) oxidase genes during the development of vegetative tissues in white clover (Trifolium repens L.) is regulated by ontological cues.

Four 5' flanking sequences, comprising the 5'-UTR and upstream promoter region, have been isolated and cloned from the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family of white clover (Trifolium repens L.), and designated TR-ACO1p, TR-ACO2p, TR-ACO3p and TR-ACO4p. Southern analysis confirmed that these sequences correspond to four distinct genes. The four corresponding genomic sequences have also been isolated and each shown to be comprised of four exons interspersed by three introns. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into white clover. Here, the TR-ACO1 5' flanking sequence directs highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the TR-ACO2 5' flanking sequence directs expression in both younger, mature green and in ontologically ageing tissue. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. The TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, but all four 5' flanking sequences directed expression in the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule, with the primacy of each promoter determined by the ontological age of the tissues. These data suggest that in vegetative tissue development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.